Anaplasma Marginale Infection Research Articles

Seroprevalence of Babesiosis and Anaplasmosis in Apparently Healthy Large Ruminants of Punjab, India

Bovine babesiosis and anaplasmosis are two important enzootic, tick-borne diseases of cattle in India. The present study reports the seroprevalence of Babesia bigemina and Anaplasma marginale infection in bovines from different district of Punjab, India. A total of 184 serum samples from six districts of Punjab were evaluated for the presence of antibodies to both the parasitic antigens using a commercially available enzyme linked immunosorbent assay kit. The overall seroprevalence of B. bigemina and A. marginale was 30.9 and 20.6 % respectively, out of which cattle (46 and 31.3 %) showed more prevalence than the buffaloes (5.8 and 2.9 %). A co-infection percentage of 14.1 (24 cattle and 2 buffaloes) were also observed. These results may serve as a support either in prevention or control of the diseases.

Loss of Immunization-Induced Epitope-Specific CD4 T-Cell Response following Anaplasma marginale Infection Requires Presence of the T-Cell Epitope on the Pathogen and Is Not Associated with an Increase in Lymphocytes Expressing Known Regulatory Cell Phenotypes.

We have shown that in cattle previously immunized with outer membrane proteins, infection with Anaplasma marginale induces a functionally exhausted CD4 T-cell response to the A. marginale immunogen. Furthermore, T-cell responses following infection in nonimmunized cattle had a delayed onset and were sporadic and transient during persistent infection. The induction of an exhausted T-cell response following infection presumably facilitates pathogen persistence. In the current study, we hypothesized that the loss of epitope-specific T-cell responses requires the presence of the immunizing epitope on the pathogen, and T-cell dysfunction correlates with the appearance of regulatory T cells. In limited studies in cattle, regulatory T cells have been shown to belong to γδ T-cell subsets rather than be CD4 T cells expressing forkhead box protein P3 (FoxP3). Cattle expressing the DRB3*1101 haplotype were immunized with a truncated A. marginale major surface protein (MSP) 1a that contains a DRB3*1101-restricted CD4 T-cell epitope, F2-5B. Cattle either remained unchallenged or were challenged with A. marginale bacteria that express the epitope or with A. marginale subsp. centrale that do not. Peripheral blood and spleen mononuclear cells were monitored for MSP1a epitope F2-5B-specfic T-cell proliferative responses and were stained for γδ T-cell subsets or CD4(+) CD25(+) FoxP3(+) T cells before and during infection. As hypothesized, the induction of T-cell exhaustion occurred only following infection with A. marginale, which did not correlate with an increase in either CD4(+) CD25(+) FoxP3(+) T cells or any γδ T-cell subset examined.

Molecular and serological in-herd prevalence of Anaplasma marginale infection in Texas cattle

Bovine anaplasmosis is an infectious, non-contagious disease caused by the rickettsial pathogen Anaplasma marginale (A. marginale). The organism has a global distribution and infects erythrocytes, resulting in anemia, jaundice, fever, abortions and death. Once infected, animals remain carriers for life. The carrier status provides immunity to clinical disease, but is problematic if infected and naïve cattle are comingled. Knowledge of infection prevalence and spatial distribution is important in disease management. The objective of this study was to assess A. marginale infection in-herd prevalence in Texas cattle using both molecular and serological methods. Blood samples from 11 cattle herds within Texas were collected and analyzed by reverse transcription quantitative real-time PCR (RT-qPCR) and a commercial competitive enzyme-linked immunosorbent assay (cELISA). Samples from experimentally infected animals were also analyzed and RT-qPCR detected A. marginale infection up to 15 days before cELISA, providing empirical data to support the interpretation of herd prevalence results. Herds with high prevalence were located in the north Texas Rolling Plains and west Trans-Pecos Desert, with RT-qPCR prevalence as high as 82% and cELISA prevalence as high as 88%. Overall prevalence was significantly higher in cattle in north and west Texas compared to cattle in east Texas (p<0.0001 for prevalence based on both RT-qPCR and cELISA). The overall RT-qPCR and cELISA results exhibited 90% agreement (kappa=0.79) and provide the first A. marginale infection prevalence study for Texas cattle using two diagnostic methods. Since cattle are the most important reservoir host for A. marginale and can serve as a source of infection for tick and mechanical transmission, information on infection prevalence is beneficial in the development of prevention and control strategies.

Clinico-pathological alterations in a case of bovine (Anaplasmosis)

A five-year-old Holstein Friesian cow was presented to Teaching Veterinary Clinical Complex, College of Veterinary Science, GADVASU, Ludhiana with a history of tick infestation and high fever. Hematological examination revealed severe Anaplasma marginale infection with reduction in hemoglobin (2 gm/dl), total erythrocyte count (1x106 cells/cu mm) and packed cell volume (6%) with absolute neutrophilia and highly activated platelets. The animal died the next day and sent for post mortem examination. Grossly the carcass was anemic and icteric. There was enlargement of spleen and petechial hemorrhages on heart and kidney. Histopathologically spleen showed lymphoid depletion and hemosiderosis. Kidney revealed tubular degeneration and necrosis besides interstitial hemorrhages. Fatty changes and mild hemorrhages besidesbiliary retention were observed in liver. Myocardial hemorrhages were also evident.

High Genetic Diversity of &lt;i&gt;Anaplasma marginale&lt;/i&gt; Detected from Philippine Cattle

ABSTRACTA total of 658 cattle in 6 provinces in the Philippines were screened for Anaplasma marginale infection by using a diagnostic heat-shock operon (groEL) gene-PCR assay. The screening-positive samples were further tested using the major surface antigen protein 1a (Msp1a) gene-PCR assay. Screening PCR results showed 130 cattle (19.8%) were positive for the A. marginale infection. Subsequent amplification using the Msp1a gene only showed 93 samples (14.1%) to be positive. In addition, 37 tandem-repeat structures, including 20 novel structures, and 41 distinct genotypes were identified. Interestingly, multiple infections of 4 different genotypes were also observed in A. marginale-infected cattle. The present study demonstrated the prevalence and characterization of diverse genotypes of A. marginale in the Philippine cattle.

Pathological observations on clinical Anaplasma marginale infection in cattle.

Gross and histopathological changes were recorded in a pregnant cattle died of clinical anaplasmosis, a tick transmitted economically important disease caused by Anaplasma marginale. Grossly emaciated carcass along with pale visible mucous membranes and pale serosal surface, splenomegaly and hepatomegaly was observed. Microscopically, in lungs variable extend of interstitial pneumonia, emphysema along with infiltration of mononuclear cells was seen. Spleen showed extensive increase in red pulp area with massive proliferation of lymphocytes. In liver marked thickening of capsule with fatty changes along with retention of bile was seen. Gall bladder showed congestion, glandular hyperplasia and thickening wall. Myocardium showed degeneration and necrosis.

Prevalence and haemato-biochemical profile of Anaplasma marginale infection in dairy animals of Punjab (India)

ObjectiveTo do the systematic comparison of prevalence of anaplasmosis by PCR and Giemsa stained thin blood smear (GSTBS) based parasitological assays in dairy cattle of Punjab, which has not been reported yet. To analyse the haematobiochemical alterations in infected animals to arrive at the conclusion regarding the pathogenicity induced by Anaplasma marginale (A. marginale) in latent and patent infection. MethodsStudy was conducted on 320 animals (236 cows, 62 calves and 22 buffaloes) of Punjab, India. PCR on genome of A. marginale was performed by targeting msp1 β gene using specific primers BAP-2/AL34S, amplifies products of size 407 bp. Questionnaires based data on the characteristics of the infected animals and management strategies of the farm were collected and correlated. ResultsHigher prevalence and more significant association was observed in the PCR based molecular diagnosis (P=0.00012) as compared to that in GSTBS (P=0.028 8) based diagnosis with various regions under study. With respect to the regions, highest prevalence was recorded in Ferozepur by PCR based diagnosis, while that in Jalandhar by GSTBS examination. Similar marked significant association of the PCR based diagnosis with the age of the animals under study (P=0.00013) was observed elucidating no inverse age resistance to A. marginale in cow calves. Haematobiochemical profile of infected animals revealed marked anemia, liver dysfunction and increase globulin concentrate indicating rise in immunoglobulin level to counteract infection. ConclusionsPCR is far more sensitive in detecting the disease even in latent infection which may act as nidus for spread of anaplasmosis to susceptible animals in endemic areas. Severity of anaemia and liver dysfunction were comparable both in patent as well as latent infection indicating pathogenicity of both.

Subclinical anaplasmosis in camel (Camelus dromedarius) and its successful therapeutic management.

On the Indian sub continent, dromedarian camel -'the ship of the desert' is an important constituent of the socio economic life style of nomadic owners in the semi arid to arid ecosystems. The animal suffers from a few parasitic diseases viz. surra, coccidiosis, sarcocystis, gastro intestinal concurrent metazoan infections, mange, nasal bots and ticks infestations. However, anaplasmosis in camel has not been reported so far from the Indian subcontinent. Systematic investigations of a 7year male Jaisalmeri camel, with a clinical history of dullness, progressive loss of condition and stamina revealed subclinical Anaplasma marginale infection. The animal had depressed haematological indices, dry and constipated bowels, pale and icteric conjunctiva suggestive of anaemia. The animal positively responded to the specific integrated therapy. Reexamination of the animal on day 21 post-therapy revealed depressed haematological indices restored to normal levels and the erythrocytes were free from the pathogen. Neglected attention, poor and/or underreporting of camel diseases vis-a-vis economic significance of the versatile animal has been discussed. This appears to be the pioneer documentation of anaplasmosis in camels from Indian subcontinent.

Molecular detection of Anaplasma marginale infection in carrier cattle

Anaplasma marginale infections are responsible for widespread morbidity and mortality particularly in crossbred and exotic breeds of cattle in the tropics and subtropics. In the present study, a semi-nested PCR assay was utilized for the detection of A. marginale infection in carrier cattle from different areas of Punjab state, India. An amplicon of 458bp specific for msp5 of A. marginale was detected in 45.2% of blood samples when subjected to primary PCR assay against the routine blood smear examination, which revealed inclusion bodies in only 12.5% of samples. Semi-nested PCR employing product of samples negative by primary PCR produced the amplicons of desired size (345bp) in 51% (29/57) of samples indicating that nested PCR, when coupled with primary PCR, resulted in increased sensitivity in detection of A. marginale infection in carrier cattle from 45.2 to 73.1%. These results suggest higher threshold detection limits of the nested PCR over the conventional technique used for diagnosis of anaplasmosis which is ideally suited for diagnosis of carrier cases which act as nidus for the spread of the infection to the susceptible stock in endemic areas.

Investigation of endothelial cells as an in vivo nidus of Anaplasma marginale infection in cattle

Continuous culture of Anaplasma marginale in endothelial cells and the potential implications for vaccine development heightened interest in determining the importance of endothelial cells in the A. marginale life cycle. A. marginale-infection trials were performed to determine if endothelial cells are an in vivo host cell in cattle and if A. marginale from in vitro endothelial cells were infective to cattle. Adult, immunocompetent steers were infected by tick-feeding transmission and were euthanized at different points in the parasitemic cycle. Based on quantitative PCR, the tissue distribution of A. marginale DNA during peak and trough parasitemia was variable with higher quantities observed in spleen, lung, hemal nodes, and abomasum. A. marginale was not conclusively identified in tissue endothelial cells from the steers’ tick-bitten dermis or post-mortem tissues using three microscopy techniques (dual indirect immunofluorescence, transmission electron microscopy, and in situ DNA target-primed rolling-circle amplification of a padlock probe). Intravenous inoculation of spleen-intact or splenectomized calves with endothelial cell culture-derived VA isolate A. marginale did not cause seroconversion or clinical anaplasmosis regardless of whether the endothelial culture-derived bacteria were inoculated as host cell-free organisms or within endothelial cells and regardless of the type of endothelial cell culture used – RF/6A primate endothelial cells or primary bovine testicular vein endothelial cells. Data presented here suggest that endothelial cells are likely not a pivotal component of the A. marginale life cycle in vivo.

Spatial variation of tick abundance and seroconversion rates of indigenous cattle to Anaplasma marginale, Babesia bigemina and Theileria parva infections in Uganda

Tick abundance and seroconversion rates of 640 indigenous cattle in a mixed crop-livestock system in Uganda were investigated in a 14 months longitudinal study. Up to 100% of the cattle in Buyimini, Kubo, Nanjeho, Ojilai and Sitengo villages (high tick challenge zone) were consistently infested with Rhipicephalus appendiculatus, whereas on average 50% of the cattle in Bunghaji, Hitunga and Magoje villages (low tick challenge zone) were inconsistently infested. Likewise, up to 50% of the cattle in Buyimini, Kubo, Nanjeho, Ojilai and Sitengo villages were consistently infested with R. (Boophilus) decoloratus ticks, while on average 30% of the cattle in Bunghaji, Hitunga and Magoje were inconsistently infested. Seroconversion rates of cattle to Anaplasma marginale infection under low tick challenge were higher than those under high tick challenge, but the reverse was true for Babesia bigemina infection. For Theileria parva infection, seroconversion rates of cattle older than 6 months under low tick challenge were significantly higher than those under high tick challenge (P < 0.05). However, the likelihood of occurrence of theileriosis cases among calves (0-6 m) under high tick challenge was 6 times (Odds ratio = 5.82 [1.30-36.37]) higher than under low tick challenge. The high density of anti-tick plants Lantana camara and Ocimum suave that were widespread in villages with low tick challenge, among other factors, was probably the cause for unfavourable tick survival.

Temporal characterisation of the organ-specific Rhipicephalusmicroplus transcriptional response to Anaplasma marginale infection

Arthropods transmit important infectious diseases of humans and animals. Importantly, replication and the development of pathogen infectivity are tightly linked to vector feeding on the mammalian host; thus analysis of the transcriptomes of both vector and pathogen during feeding is fundamental to understanding transmission. Using Anaplasma marginale infection of Rhipicephalusmicroplus as the experimental model, we tested three hypotheses exploring the temporal and organ-specific nature of the tick midgut and salivary gland transcriptomes during feeding and in response to infection. Numerous R. microplus genes were regulated in response to feeding and were differentially regulated between the midgut and salivary gland; additionally, there was a progression in regulated gene expression in the salivary gland over time. In contrast, relatively few tick genes were specifically regulated in response to A. marginale infection and these genes were predominantly annotated as hypothetical or were of unknown function. Notable among the genes with informative annotation was that several ribosomal proteins were down-regulated, suggesting that there may be a corresponding decrease in translation. The hypotheses that R. microplus midgut and salivary gland genes are differentially regulated and that the salivary gland transcriptome is dynamic over time were accepted. This is consistent with, and important for understanding the roles of, the two organs, the midgut serving as an initial site of uptake and replication while the salivary gland serves as the final site of replication and secretion. The nominal effect of A. marginale on the tick transcriptome in terms of numbers of regulated genes and fold of regulation supports the view that the vector–pathogen relationship is well established with minimal deleterious effect on the tick. The small set of predominantly hypothetical genes regulated by infection suggests that A. marginale is affecting a novel set of tick genes and may provide new opportunities for blocking transmission from the tick.

The efficacy of three chlortetracycline regimens in the treatment of persistent Anaplasma marginale infection

Chemosterilization is reported in cattle fed chlortetracycline hydrochloride (CTC) at dosages ranging from 1.1 mg/kg for 120 days to 11 mg/kg for 30–60 days. The relationship between plasma CTC drug concentration and carrier clearance has not been described. Chronic carrier status was established in 21 steers with a Virginia isolate of Anaplasma marginale and confirmed by cELISA and an A. marginale-specific RT-PCR. Four negative, splenectomized steers served as active disease transmission sentinels. Steers were randomized to receive 4.4 mg/kg/day (LD); 11 mg/kg/day (MD); or 22 mg/kg/day (HD) of oral chlortetracycline; or placebo (CONTROL) for 80 days. The LD, MD and HD treatment groups consisted of 5 infected steers and 1 splenectomized steer; CONTROL group had six infected steers and 1 splenectomized steer. The daily treatments and ration were divided equally and fed twice daily. Blood samples were collected semi-weekly for determining plasma drug concentration by ultrahigh performance liquid chromatography–mass spectrometry/mass spectrometry method and assessment of disease status by both cELISA and RT-PCR. Mean (CV%) chlortetracycline plasma drug concentrations in the LD, MD, and HD groups were 85.3 (28%), 214.5 (32%) and 518.9 (40%) ng/mL during days 4 through 53 of treatment. A negative RT-PCR assay result was confirmed in all CTC-treated groups within 49 days of treatment; however, cELISA required an additional 49 to 88 days before similar results. Subinoculation of splenectomized steers confirmed chemosterilization. These results are important for influencing future chemosterilization strategies and impacting free trade policy among countries and regions of contrasting endemicity.

Anaplasma marginale infection in cattle from south-western Amazonia

The present study provides the first epidemiological data regarding infection by Anaplasma marginale in cattle reared in south-western Brazilian Amazonia. One simple procedure was adapted for the extraction of DNA from blood clots collected in seven microregions of Rondônia State and two mesoregions of Acre State. PCR method was used to asses the frequency of A. marginale infections in 4 to12-month-old cattle. The cattle infection was investigated by polymerase chain reaction (PCR) using the specific primer "msp5" for A. marginale. The DNA amplifications revealed that the mean frequency of A. marginale infection was 98.6% (1,627/1,650) in samples from Rondonia, and 92.87% (208/225) in samples from Acre. The high frequency of A. marginale infections in 4 to 12-month-old cattle indicate a situation of enzootic stability in the studied areas and are comparable to those detected by immunodiagnosis in different endemic regions in Brazil. The DNA extraction of clotted blood method described here can be used for epidemiological studies on anaplasmosis and other bovine hemoparasites.

Diagnostic Accuracy of Methods for Detecting Anaplasma Marginale Infection in Lactating Dairy Cattle of Puerto Rico

Bovine anaplasmosis (BA) is a hemoparasitic disease of great importance in cattle within the tropical and subtropical regions of the world. Control programs for BA require accurate diagnostic assays but validation can be challenging because the true disease status of all animals is frequently not known with certainty. The objective of this study was to estimate the accuracy of assays for detection of Anaplasma marginale infection in lactating dairy cattle of Puerto Rico using Bayesian methods without a perfect reference test. There were 2,331 cattle with complete diagnostic results sampled from 79 herds, and the prevalence of BA was estimated as 22% (95% probability interval [PI]: 19-25%). The sensitivity (Se) and specificity (Sp) of a major surface protein 5 competitive enzyme-linked immunosorbent assay (MSP-5 cELISA) were estimated as 99% (95% PI: 96-100%) and 89% (95% PI: 87-92%), respectively. The Se and Sp of a quantitative polymerase chain reaction (qPCR) were 67% (95% PI: 60-74%) and 99% (95% PI: 99-100%). The Se and Sp of a card agglutination test were 34% (95% PI: 29-39%) and 99% (95% PI: 99-100%). Area under the receiver-operating characteristic curve for the MSP-5 cELISA was 0.748 (95% PI: 0.71-0.79). The MSP-5 cELISA appears to be the test of choice for screening cattle for subclinical BA based on the high estimated Se, rapidity of results, relative low cost, and ease of standardization.

Comparison of Three Tetracycline Antibiotic Treatment Regimens for Carrier Clearance of Persistent Anaplasma marginale Infection Derived under Field Conditions

Anaplasmosis, caused by the intracellular red blood cell parasite Anaplasma marginale, is the most prevalent tick-transmitted disease of cattle worldwide. Cattle recovering from acute anaplasmosis, including those treated with tetracycline antibiotics, develop lifelong cyclic rickettsemias. Carrier cattle serve as a reservoir of A. marginale for the infection of naïve cattle through horizontal, vertical, and iatrogenic transmission. Validated antimicrobial regimens do not exist for eliminating persistent infection; furthermore, anaplasmosis disease control is confounded by the absence of effective vaccines. A diagnostic testing strategy for identification of A. marginale infection and determination of chemotherapeutic success is urgently needed. A novel, real time RTPCR diagnostic assay and oral chlortetracycline (CTC) chemosterilization strategy were developed at Kansas State University under experimental conditions through concurrent RT-PCR results and plasma CTC pharmacokinetic determination. Validation of this diagnostic and treatment strategy utilizing cattle naturally-infected is essential for assembling science-based recommendations for anaplasmosis control, treatment, and eradication.

Comparative moleculo-immunological analysis of swamp- and riverine-type water buffaloes responses

This moleculo-epidemiological and immunological study through cytokine response assessment was done to know the dynamics of cytokines in the initiation, persistence and association to physiological changes of a particular pathogen in water buffaloes. This is important to understand the magnitude and behavior of disease progression. Water buffalo blood samples gathered from different places in the Philippines revealed a 9.4%, 27.6%, 10.3% and 4.4% prevalence of bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), Anaplasma marginale and Babesia bigemina infection, respectively. This was the first surveillance study of BVDV and BLV in the country. Furthermore, cytokine expression of these naturally infected animals was also quantified. BVDV-infected animals had up-regulated expressions of TNFα, IL-2 and IL-4; and down-regulated expressions of IFNγ and IL-12p40 while BLV positive animals had an up-regulated IL-4 and IL-6, and highly expressed IL-10 and IL-12p40 with unchanged IFNγ expression. Meanwhile, animals infected with A. marginale had all interleukins and IFNγ up-regulated with significant expression of IL-10 and IL-12p40 similar to the BLV positive animals. Since it was also observed that swamp-type buffaloes were more disease tolerant than riverine-type buffaloes based on the gathered infection rate of each examined pathogen, further assessment was done focusing on the two vital cytokines, IFNγ and TNFα. We quantified IFNγ and TNFα expressions in ConA-stimulated PBMC from both swamp and riverine buffaloes by real-time PCR. Cytokine expression from ConA-stimulated PBMC revealed that both IFNγ and TNFα were more highly expressed in swamp than in riverine buffalo. To further examine the probable cause of expression differences, the proximal promoter region of these two cytokines were sequenced for the presence of nucleotide polymorphism followed by luciferase assay to analyze the effect of these polymorphisms in gene transcription. A single nucleotide polymorphism was found in the IFNγ (−299) while eight polymorphisms in the TNFα promoter (−541, −553, −562, −596, −609, −655, −659, −688). Luciferase assay showed that both IFNγ promoter and TNFα promoter in swamp-type water buffalo had higher transcription activity compared to riverine-type water buffalo. These findings confirm that IFNγ and TNFα transcriptions in these animals were highly affected by the disparity in the cytokine promoter region. This suggests that disease tolerance or susceptibility of these buffaloes could be due to the differences in their relative cytokine transcription and may relate to pathogen–host specific pathogenesis.

Anaplasma marginale infection in a Japanese Black cow 13 years after eradication of Rhipicephalus ( Boophilus) microplus in Okinawa, Japan

In October 2007, a 15-year-old Japanese Black cow on Ishigaki Island, Okinawa, Japan, was diagnosed with Anaplasma marginale infection based on clinical symptoms, blood examination, smear observation, 16S rRNA and groEL gene sequence analysis, and the result of a CF test. The cow was introduced into the farm from mainland Japan as a calf in 1993, one year before the eradication of Rhipicephalus ( Boophilus) microplus, the main vector of A. marginale in Okinawa Prefecture. It is possible that the cow was first infected with A. marginale as a calf in Ishigaki Island and had been persistently infected since then. This is the first reported clinical case of A. marginale infection of cattle since the eradication of R. microplus in Okinawa Prefecture. Additional analysis of major surface protein 1α amino acid sequences revealed that the A. marginale Okinawa strain presented four new repeat forms which were not seen in other strains. This indicates that the Okinawa strain may be a unique geographical variant of A. marginale.

Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections

Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections.

Validation of an Anaplasma marginale cELISA for use in the diagnosis of A. ovis infections in domestic sheep and Anaplasma spp. in wild ungulates

A commercially available (cELISA) kit for diagnosing Anaplasma marginale infection in cattle was validated for diagnosing A ovis infection in sheep using the bovine serum controls as supplied by the manufacturer (BcELISA) and sheep serum controls from pathogen-free sheep (OcELISA). True positives were identified using two previously established assays, a nested PCR (nPCR) test and an indirect immunofluorescent assay (IFA). The BcELISA was also applied to sera from various species of wild ruminants, comparing the results with the IFA. Receiver operating characteristic (ROC) analysis indicated that the predicted threshold inhibition for the BcELISA was 19.2. The sensitivity for the BcELISA was 98.2% and the specificity was 96.3%. The predicted threshold inhibition decreased to 14.3 for the OcELISA; the sensitivity was 96.5% and the specificity was 98.1%. There was ≥90% concordance between IFA and nPCR, as well as between the BcELISA at 19% inhibition cutoff and either IFA or PCR. Concordance between the cELISA and IFA using sera from elk, mule deer, bighorn sheep, pronghorn antelope, and black-tailed deer ranged from 64% to 100%. This commercially available cELISA test kit can be used very effectively to test domestic sheep for infection with A. ovis using the kit-supplied controls (i.e. the BcELISA) and a 19% inhibition cutoff; the kit may also be useful for detecting intra-erythrocytic Anaplasma infections in wild ruminants.